IgE mediates, among other things, immediate-type hypersensitivity reactions. For an allergic reaction to occur, an individual must have had prior exposure to an allergen. Following the initial antigen exposure, the immune system produces IgE specific for the inciting antigen. The antigen-specific IgE then binds to mast cell membranes via IgE receptors. When re-exposed to the antigen, the antigen-specific IgE antibody binds to the antigen and activates the mast cells. Such mast cell activation causes a release of vasoactive and neuronal stimulatory mediators such as histamines, leukotrienes, prostaglandins, bradykinin, and platelet-activating factor which work in conjunction with cells such as eosinophils, basophils, neutrophils, and CD4 T-lymphocytes. Allergen induced IgE secretion can result in a variety of complications, including death, as may be the case in serious cases of asthma and anaphylaxis. These allergic disorders are prevalent. For example, allergic rhinitis (hay fever) affects 22% or more of the population of the USA, whereas allergic asthma is thought to affect at least 20 million residents of the USA. The economic impact of allergic diseases in the United States, including health care costs and lost productivity, has been estimated to amount to $6.4 billion in the early nineties alone.
IgE is secreted by IgE-producing plasma cells, which differentiate from B cells expressing membrane-bound IgE (mIgE) on their surface. IgE not only has the shortest biologic half-life of all classes of immunoglobulins (Igs), but also is present in serum at the lowest levels. However, IgE concentrations in allergic reactions (atopic) in individuals can be 100- to 1000-fold higher than in normal individuals. IgE is directly involved in mediating many allergic reactions as a result of its ability to bind to and, upon contact with multivalent allergen, activate various effector cells, such as mast cells and basophils, through interactions with FcεRI receptors.
Since IgE plays a central role in mediating most allergic reactions, devising treatments to control IgE levels in the body and regulating IgE synthesis has been of great interest. Several strategies have been proposed to treat IgE-mediated allergic diseases by downregulating IgE levels. One strategy involves neutralizing the IgE molecules by binding the ε-chain of IgE in or near the Fc-receptor binding site. For example, Omalizumab (Xolair) is a recombinant humanized monoclonal anti-IgE antibody that binds to IgE on the same Fc site as FcεRI. Omalizumab causes a reduction in total serum IgE in atopic patients, which attenuates the amount of antigen-specific IgE that can bind to and sensitize tissue mast cells and basophils. This, in turn, leads to a decrease in symptoms of allergic diseases.
While Omalizumab reduces the amount of free IgE (the unbound form present in the circulation) it does not bind to IgE already bound to effector cells nor does it bind to membrane-anchored IgE. Thus, while neutralizing anti-IgE antibodies, like Omalizumab, may reduce the severity of some IgE-mediated allergic diseases they may not be effective for treating patients with very high levels of soluble IgE. Nor will they likely be effective for the treatment of diseases caused by monoclonal expansion of B-cells, such as, Job's disease. Strategies to treat these diseases focus on depleting the B-cells producing IgE for example, by binding membrane-anchored IgE present on the surface of B-cells and targeting these cells for destruction by a variety of mechanisms including the use of cytotoxic agents and mediating cell killing pathways such as antibody dependent cell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). These methods would be efficacious for both the treatment of IgE-mediated allergic disease as well as for disease caused by the expansion of IgE expressing B-cells. Furthermore, these methods could be adapted to treat other diseases caused by monoclonal expansion of B-cells expressing other membrane-anchored immunoglobulins such as, for example, IgM expressing B-cells in Waldenstrom Macroglubulinemia, IgA and IgG expressing B-cells in various myelomas and autoimmune diseases and IgM and IgA expressing B-cells in neuropathy and nephropathy, post transplant lymphoproliferative disorder (PTLD), and monocolonal gammopathy of unknown significance (MGUS).
There are two forms of immunoglobulins: the secreted and the membrane anchored form. The membrane-anchored form differs from the secreted form in that the former has a membrane-anchoring peptide extending from the C terminus of the heavy-chain. Membrane-anchored immunoglobulin on B-cells is critical for B-cell functions. It can transduce signals for resting B cells to differentiate into activated lymphoblasts and Ig-secreting plasma cells. The amino acid sequences of many membrane-anchored immunoglobulins are known. These sequences share certain common features including the presence of a membrane anchoring peptide. The membrane anchoring peptide has three segments that are distinguishable based on their locations in relation to the plasma membrane (extracellular segment, transmembrane segment, and cytoplasmic segment). The N-terminal segment (extracellular segment) of the anchoring peptides is often designated as hydrophilic and highly acidic. This segment can be easily identified by amino acid sequence comparison and analysis and is referred to as the membrane-anchored immunoglobulin isotype specific (“migis”) peptide or epitope (see FIG. 1A).
The migis peptides are unique for the different immunoglobulin isotypes. Therefore, the extracellular segment of the ε-chain membrane anchoring peptide forms, in whole or in part, an epitope unique to the B cells which produce IgE. The same is true for each immunoglobulin isotype. Furthermore, the migis peptide is not present on secreted, soluble immunoglobulin because only the immunoglobulin which is bound to the surface of B cells contains the membrane anchoring peptide as part of its heavy chain. Thus, therapeutics which specifically targeted the migis peptides would be useful to target specific classes of B-cells for the treatment of a wide variety of conditions including allergic diseases and those mediated by monoclonal B-cell expansion.
Membrane anchored IgE is found in at least two isoforms as a result of alternative splicing in humans. The ε-chain of both isoforms of human mIgE contains a ε-migis epitope and a membrane-anchoring peptide. One isoform contains only the ε-migis sequence (a 15-amino-acid-long domain) between the membrane anchor sequence and the C4 region, referred to as the short form. Whereas, the second isoform additionally contains an extra 52-amino-acid (a.a.)-long domain, referred to as cεmx, between the CH4 domain and the ε-migis sequence, referred to as the long form (see FIG. 2). Several groups have generated mouse monoclonal antibodies that bind to either the ε-migis peptide (see, e.g., Chang et al. U.S. Pat. Nos. 5,422,258 and 5,091,313) or an 8 amino acid cεmx peptide (Chen et al. 2002, Int Arch Allergy Immunol 128:315-24). However, as demonstrated herein (see Section 6, Example 1), antibodies that recognize the ε-migis peptide alone are likely to cross react with another commonly expressed cell surface protein, while those which interact with a predominant epitope present on the 8 amino acid cεmx peptide may in fact be hidden when the immunoglobulin is present on the membrane. Furthermore, it is not desirable to use mouse antibodies directly as a human therapeutic due to the generation of human-anti-mouse antibodies (HAMA) or HAMA response. Thus, antibodies of non-human origin are preferably engineered to “humanize” them to prevent eliciting a HAMA response. The process of humanization is not only time consuming but often results in an antibody with altered binding characteristics that is not a useful therapeutic. The antibodies disclosed herein are fully human antibodies which bind a unique ε-chain migis epitope not previously described.
Citation or discussion of a reference herein shall not be construed as an admission that such is prior art to the present invention.